Abstract: Lung transplantation (LT) is the only therapeutic option for patients with end-stage lung disease; however, survival lags behind other organ transplants, and is primarily due to chronic lung allograft dysfunction (CLAD), most often the bronchiolitis obliterans syndrome (BOS). Episodes of acute cellular rejection (ACR) are common and the major risk factor for CLAD, despite calcineurin inhibitor-based (e.g., cyclosporine; CSA) conventional immunosuppression therapy (IST). Experimental transplant models and humans studies indicate that Type-1 T cell immunity, marked by IFN-? production, and driven by the transcription factor, T-bet, play a key role in allograft rejection. Thus, new and effective therapeutic strategies represent a major unmet need in LT and are desperately needed to prevent lung rejection and improve outcomes. We have discovered a novel, pro-inflammatory ubiquitin, E3 ligase subunit, Fbxo3, which degrades an anti-inflammatory calmodulin (CaM)- associated protein called Fbxl2. Our preliminary data show Fbxo3 is induced during ACR in the mouse orthotopic lung transplant (mouse OLT) model, along with T-bet, while Fbxl2 is markedly reduced. Further, using a first-in-class novel small molecule inhibitor of Fbxo3, BC-1261, reduces T-bet/Type-1 immunity and ACR severity, and preserves Fbxl2 in our early studies. Our studies in lung transplant recipients (LTRs) show a predominance of Type-1 alloimmune responses in the lung and blood, up-regulation of Fbxo3, and inhibition of immune responses with BC-1261. Therefore, we hypothesize that Fbxl2 is a critical regulator of T-bet/Type-1 immunity in T cells, and that Fbxo3: Fbxl2 balance and T-bet/IFN-? induction are key determinants of lung allograft rejection versus acceptance. To test this, we propose 3 aims. In Aim 1, we will test the hypothesis that induction of Fbxo3 degrades Fbxl2 and increases T-bet in activated T cells. We will determine whether Fbxl2 ubiquitinates T-bet, whether CaM acts as a linker molecule in regulating T-bet, and test the regulation of Fbxo3 during T cell activation. In Aim 2, using the mouse OLT model, we will test the hypothesis that the balance of functional Fbxo3: Fbxl2 and T-bet/Type-1 immunity are key determinants of lung allograft outcomes. Here, we will assess our novel Fbxo3 inhibitor, BC-1261 versus CSA on ACR or obliterative airways disease (OAD; modeling BOS), and test whether lung rejection is reduced in novel Fbxo3-deficient mice. In Aim 3, we will test the hypothesis that the Fbxo3 and T-bet/Type-1 immunity pathways are putative targets in human LT and whether BC-1261 inhibits alloimmune responses. Making use of our expanding LTR registry/biorepository of bronchoalveolar lavage (BAL) and PBMC samples, we will determine whether F-box proteins play an important role in lung allograft and systemic alloimmunity. The PIs, Drs. McDyer and Chen, bring multidisciplinary expertise combining ubiquitin biology, mouse and human lung transplant immunology, and will use the multiple PI format. Success in this R01 will be transformative in the science to prevent lung allograft rejection, and lay the foundation to test a first-in-class therapy targeting the pro-inflammatory ubiquitin, E3 ligase subunit, Fbxo3.